Identification and primary application of hybridomas cell secreting monoclonal antibodies against mink (Neovison vison) interferon-gamma.

Due to their susceptibility to several human viruses, the mink has been proposed as potential animal models for the study of human viral infections. However, there are no specific monoclonal antibody (mAbs) currently available for the detection of mink-specific interferon-gamma (miIFN-γ). The BALB/c mice were immunized intraperitoneally with purified recombinant miIFN-γ protein. The splenocytes were obtained and fused with murine myeloma cells. Five of 24 hybridoma clones were obtained to produce mAbs steadily with the strongest affinity to recombinant miIFN-γ protein. The isotype of the 31A, 31B and 31G were lgG 2b. The isotype of 44 and 46 were lgG 2a and 1. All five mAbs were κ light chains. Western blotting and indirect ELISA method showed that 5 mAbs were positive to miIFN-γ. Immunofluorescence showed that 2 mAbs (44 and 46) had a positive reaction to miIFN-γ. The hybridoma clone 46 had the highest sensitivity for the detection of miIFN-γ. Most importantly, our primary sandwich ELISA system (mAbs 46 and polyclonal antiserum) detected endogenous IFN-γ in mink lymphocytes infected with canine distemper virus (CDV). We have thus developed a novel mAbs could recognize miIFN-γ, and have demonstrated the first ELISA-based measurement of IFN-γ in lymphocyte of the mink.

LTM-TCM: A comprehensive database for the linking of Traditional Chinese Medicine with modern medicine at molecular and phenotypic levels.

Benefiting from the development of network pharmacology, Traditional Chinese Medicine (TCM) shows great potential in modern drug discovery. Recently, more and more TCM-related databases have been established for both academic and industry research, but they are still insufficient in data standardization, integrity, and precision. To better accelerate the TCM research and overcome these shortcomings, we construct a web-based TCM platform, LTM-TCM, which is currently the most comprehensive TCM database that includes the following advantages: (1) High-quality data integration from fourteen TCM authoritative databases, especially with additional manual collected 41,025 clinical treatment records and 213 ancient Chinese medical books. (2) Accurate correction of multi-source TCM interactions (between symptoms, prescriptions, herbs, ingredients and targets) through in-house Biomedical Natural Language Processing (BioNLP) approaches in more than 30 million articles. (3) Diverse cross-field pipelines (e.g., bioactive ingredients screening, targets prediction, and mechanism prediction, etc.) help integrating traditional medicine with modern science in common aspects at both the molecular and phenotypic levels. In summary, LTM-TCM contains 1928 symptoms, 48,126 prescriptions, 9122 plants, 34,967 ingredients, 13,109 targets and 1170,133 interactions among all TCM related components. LTM-TCM has both Chinese and English interfaces, and it is accessible at http://cloud.tasly.com/#/tcm/home.

The biological characteristics of the canine adenovirus type 1 from fox and the transcriptome analysis of the infected MDCK cell.

Canine adenovirus type 1 (CAdV-1) is the etiologic agent of fox encephalitis, and a virus strain from fox encephalitis is isolated and related research are conducted. In this experiment, the results showed that the F1301 strain was confirmed to be the CAdV-1. The whole genome of the CAdV-1 F1301 strain isolated from fox was 30,535 bp and had higher homology to the other reported CAdV-1 strains. After 0, 12, and 36 h of CAdV-1 infection, the difference gene of the 592 long noncoding RNA and 11,215 microRNA were involved in cell responses to CAdV-1 infection through the PI3K-AKT, Wnt, Herpes simplex, hepatitis C, and Epstein-Barr virus infection pathway in Madin-Darby canine kidney cell line (MDCK). The results indicate that the biological characterization of the CAdV-1 and the MDCK cell-CAdV-1 interaction are clarified.

Compound Danshen Dripping Pill inhibits high altitude-induced hypoxic damage by suppressing oxidative stress and inflammatory responses.

CONTEXT: Previous studies indicate that compound Danshen Dripping Pill (CDDP) improves the adaptation to high-altitude exposure. However, its mechanism of action is not clear. OBJECTIVE: To explore the protective effect of CDDP on hypobaric hypoxia (HH) and its possible mechanism. MATERIALS AND METHODS: A meta-analysis of 1051 human volunteers was performed to evaluate the effectiveness of CDDP at high altitudes. Male Sprague-Dawley rats were randomized into 5 groups (n = 6): control at normal pressure, model, CDDP-170 mg/kg, CDDP-340 mg/kg and acetazolamide groups. HH was simulated at an altitude of 5500 m for 24 h. Animal blood was collected for arterial blood-gas analysis and cytokines detection and their organs were harvested for pathological examination. Expression levels of AQP1, NF-κB and Nrf2 were determined by immunohistochemical staining. RESULTS: The meta-analysis data indicated that the ratio between the combined RR of the total effective rate and the 95% CI was 0.23 (0.06, 0.91), the SMD and 95% CI of SO2 was 0.37 (0.12, 0.62). Pre-treatment of CDDP protected rats from HH-induced pulmonary edoema and heart injury, left-shifted oxygen-dissociation curve and decreased P50 (30.25 ± 3.72 vs. 37.23 ± 4.30). Mechanistically, CDDP alleviated HH-reinforced ROS by improving SOD and GPX1 while inhibiting pro-inflammatory cytokines and NF-κB expression. CDDP also decreased HH-evoked D-dimer, erythrocyte aggregation and blood hemorheology, promoting AQP1 and Nrf2 expression. DISCUSSION AND CONCLUSIONS: Pre-treatment with CDDP could prevent HH-induced tissue damage, oxidative stress and inflammatory response. Suppressed NF-κB and up-regulated Nrf2 might play significant roles in the mechanism of CDDP.

Molecular characteristics of the capsid protein VP2 gene of canine parvovirus type 2 amplified from raccoon dogs in Hebei province, China.

Canine parvovirus type 2 (CPV-2) is currently circulating in domestic and wild animals, but our knowledge about CPV-2 infections in raccoon dogs is limited. In this study, VP2 gene sequences of CPV-2 were amplified from rectal swabs of 14 diarrhetic raccoon dogs (Nyctereutes procyonoides) in Hebei province, China, in 2016 and 2017. Phylogenetic analysis of the VP2 gene sequences revealed that most of these sequences (11 of 14) belonged to the same subclade as raccoon dog strain CPV-2/Raccoon_Dog/China/DP-1/16 isolated from Shandong province in 2016. A comparison of deduced amino acid sequences revealed presence of the substitutions S297A and S27T in 11 of those 14 sequences. I418T was observed in a minority of the sequences (4 of 14). In addition, A300D and T301I, P13S and I219V, and N419K were found in three of the sequences. This study shows that CPV-2 strains with different substitutions in their VP2 amino acid sequences were spreading among raccoon dogs in Hebei during 2016 and 2017 and suggests that further studies are needed to monitor the distribution of these strains in China.

[Study of intelligent manufacturing quality digitalization of traditional Chinese medicine and practice on Compound Danshen Dripping Pills].

China healthcare industry has gradually developed the consumer-centric integrated service model. To satisfy consumers' increasing demands on pluralistic, personalized and transparent healthcare services, pharmaceutical manufacturing enterprises must provide high-quality, precise and flexible medicines. This can be achieved by accelerating implementation of intelligent manufacturing, which is the core competitiveness of pharmaceutical manufacturing enterprises. According to the authors' intelligent manufacturing projects in a traditional Chinese medicine(TCM) factory, study and industrial practice on intelligent manufacturing were presented in this paper. First, the quality digitalization-based intelligent manufacturing methodology of TCM was proposed in this paper. The methodology mainly included three digitalized technologies in process and quality design, manufacturing process control and product batch evaluation. Next, the architectural design of intelligent manufacturing systems in one TCM factory was introduced, and the functional modules and data transmission relationships covering seedling, cultivation, herbal slices, preparation, storage and quality management systems were described. Finally, these technologies were fully used, and an integrated quality digitalization system was successfully established in the production workshop of a TCM product Compound Danshen Dripping Pills. The actual operation and application of process analyzers, supervisory control and data acquisition(SCADA), manufacturing execution system(MES), data analysis system, and enterprise resource planning system(ERP) were introduced. This paper provides reference for technical path planning and systematic architecture of TCM intelligent manufacturing.

Highly Pathogenic Swine Getah Virus in Blue Foxes, Eastern China, 2017.

We isolated Getah virus from infected foxes in Shandong Province, eastern China. We sequenced the complete Getah virus genome, and phylogenetic analysis revealed a close relationship with a highly pathogenic swine epidemic strain in China. Epidemiologic investigation showed that pigs might play a pivotal role in disease transmission to foxes.

Contribution of the serine protease HtrA in Escherichia coli to infection in foxes.

Escherichia coli can cause severe, acute hemorrhagic pneumonia and systemic infection in farmed foxes, raccoon dogs and minks, leading to considerable economic losses to the farmers. It is well established that the htrA-encoded serine protease HtrA is critical for bacterial growth and survival under stress, and HtrA has been determined to be a potential vaccine target. However, the roles of HtrA in E. coli pathogenesis remain unknown. In this study, we generated an htrA-deletion mutant of the E. coli protype strain HBCLE-12 that causes pneumonia in silver foxes and then evaluated the changes in bacterial physiological characteristics in the absence of HtrA. The data show that knockout of the htrA gene did not affect growth and biochemical characteristics but led to impaired virulence of the strain. Increased susceptibility to environmental stresses, impaired survival in serum, and reduced biofilm formation may contribute to the virulence attenuation of the mutant. Furthermore, the HtrA-deficient mutant was subjected to RNA-seq analysis, and 16 differentially expressed genes were determined. This study provided insight that HtrA plays a definitive role in E. coli-induced infection.

A multiplex PCR method for the simultaneous detection of three viruses associated with canine viral enteric infections.

The aim of this study was to establish a multiplex PCR (mPCR) method that can simultaneously detect canine parvovirus (CPV-2), canine coronavirus (CCoV) and canine adenovirus (CAV), thereby eliminating the need to detect these pathogens individually. Based on conserved regions in the genomes of these three viruses, the VP2 gene of CPV-2, the endoribonuclease nsp15 gene of CCoV, and the 52K gene of CAV were selected for primer design. The specificity of the mPCR results showed no amplification of canine distemper virus (CDV), canine parainfluenza virus (CPIV), or pseudorabies virus (PRV), indicating that the method had good specificity. A sensitivity test showed that the detection limit of the mPCR method was 1 × 104 viral copies. A total of 63 rectal swabs from dogs with diarrheal symptoms were evaluated using mPCR and routine PCR. The ratio of positive samples to total samples for CPV-2, CCoV, and CAV was 55.6% (35/63) for mPCR and 55.6% (35/63) for routine PCR. Thirty-five positive samples were detected by both methods, for a coincidence ratio of 100%. This mPCR method can simultaneously detect CCoV (CCoV-II), CAV (CAV-1, CAV-2) and CPV-2 (CPV-2a, CPV-2b, CPV-2c), which are associated with viral enteritis, thereby providing an efficient, inexpensive, specific, and accurate new tool for clinical diagnosis and laboratory epidemiological investigations.

Development of a TaqMan probe-based quantitative reverse transcription PCR assay for detection of Getah virus RNA.

Getah virus (GETV), a mosquito-borne virus that mainly infects horses and pigs, has emerged and spread in China. We developed a highly specific and reproducible TaqMan probe-based quantitative reverse transcription PCR (RT-qPCR) assay targeting the non-structural protein 1 of GETV, whose detection limit is 25.5 copies/µL, which is 100-fold higher than that of conventional RT-PCR. RT-qPCR was used to detect GETV RNA in mosquito and animal clinical samples, showing that the accuracy of RT-qPCR was higher than that of conventional RT-PCR. The newly developed RT-qPCR assay may be a useful alternative tool for rapid, simple and specific diagnosis of GETV infection.

Outbreak of severe pseudorabies virus infection in pig-offal-fed farmed mink in Liaoning Province, China.

An outbreak of severe pseudorabies virus (PRV) infection in farmed mink occurred in northern China in late 2014, causing significant economic losses in the local fur industry. Here, we report the first case of a PRV outbreak in mink in northeastern China, caused by feeding farmed mink with raw pork or organs contaminated by PRV. Mink infected with virulent PRV exhibited diarrhea, neurologic signs, and higher mortality, which can be misdiagnosed as highly pathogenic mink enteritis virus (MEV), canine distemper virus (CDV), and food poisoning. However, these were excluded as causative agents by PCR or bacteria isolation. The duration of disease was 3-7 days, and the mortality rate was 80-90%. PRV was characterized using indirect immunofluorescence assays (IFA) and electron microscopy (EM). Phylogenetic analysis based on full-length genome sequences and those of individual genes of this novel virus strain showed that it clustered in an independent branch with several other PRV isolates from China.

High-performance liquid chromatography based chemical fingerprint analysis and chemometric approaches for the identification and distinction of three endangered Panax plants in Southeast Asia.

Among Panax genus, only three endangered species Panax notoginseng, P. vietnamensis, and P. stipuleanatus that have a similar morphology are mainly distributed in Southeast Asia. These three plants are usually misidentified or adulterated. To identify them well, their chemical chromatographic fingerprints were established by an effective high-performance liquid chromatography method. By comparing the chromatograms, the three Panax species could be distinguished easily using the 22 characteristic peaks. Besides, the data of the chromatographic fingerprints aided by chemometric approaches were applied for the identification and investigation the relationship of different samples and species. Using similarity analysis, the chemical components revealed higher similarity between P. vietnamensis and P. stipuleanatus. The results of hierarchical clustering analysis indicated that samples belonging to the same species could be clustered together. The result of principal component analysis was similar with hierarchical clustering analysis and the three principal components accounted for >80.5% of total variability.

Cloning, expression and antiviral activity of mink alpha-interferons.

BACKGROUND: As a key link between innate and adaptive immune responses, the interferon (IFN) system is the first line of defense against viral infection. IFN, and in particular, IFN-α, has been used clinically as an effective therapeutic agent for viral infections. However, different subtypes of IFN-α demonstrate distinct antiviral activity. Therefore, it is important to identify IFN-α subtypes with high antiviral activity for the development of genetically engineered antiviral drugs. RESULTS: In this study, we cloned the genes for 13 IFN-α subtypes from peripheral blood lymphocytes of the mink. The homologies of the 13 mink IFN-α genes were 93.6-99.3% and 88.8-98.4% at the nucleotide and amino acid sequence levels, respectively. In contrast to human and canine IFN-α subtypes, most mink IFN-α subtypes contained two N-glycosylation sites. We expressed and purified 13 mink IFN-α subtypes in Escherichia coli. The cytopathic effect inhibition assay showed that all the 13 recombinant mink IFN-α subtypes inhibited the propagation of vesicular stomatitis virus in WISH cells, with IFN-α2 and IFN-α12 demonstrating the highest activities. Furthermore, recombinant mink IFN-α2 and IFN-α12 significantly suppressed the propagation of canine distemper virus in Vero cells, with IFN-α2 demonstrating the highest activity. CONCLUSIONS: We identified the mink IFN-α2 subtype as a promising candidate for the development of effective antiviral drugs.

Nitric Oxide Plays a Central Role in Water Stress-Induced Tanshinone Production in Salvia miltiorrhiza Hairy Roots.

Nitric oxide (NO), a well-known signaling molecule plays an important role in abiotic and biotic stress-induced production of plant secondary metabolites. In this study, roles of NO in water stress-induced tanshinone production in Salvia miltiorrhiza hairy roots were investigated. The results showed that accumulations of four tanshinone compounds in S. miltiorrhiza hairy roots were significantly stimulated by sodium nitroprusside (SNP, a NO donor) at 100 µM. Effects of SNP were just partially arrested by the mevalonate (MVA) pathway inhibitor (mevinolin), but were completely inhibited by the 2-C-methyl-d-erythritol-4-phosphate pathway (MEP) inhibitor (fosmidomycin). The increase of tanshinone accumulation and the up-regulation of HMGR and DXR expression by PEG and ABA treatments were partially inhibited by an inhibitor of NO biosynthesis (Nω-nitro-L-arginine methyl ester (L-NAME)) and a NO scavenger (2-(4-Carboxyphenyl)- 4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO)). Simultaneously, NO generation in the hairy roots was triggered by PEG and ABA, and the effects were also arrested by c-PTIO and L-NAME. These results indicated that NO signaling probably plays a central role in water stress-induced tanshinone production in S. miltiorrhiza hairy roots. SNP mainly stimulated the MEP pathway to increase tanshinone accumulation.

Isolation and complete nucleotide sequence of a Batai virus strain in Inner Mongolia, China.

BACKGROUND: Batai virus (BATV) is a member of the Orthobunyavirus genus of the family Bunyaviridae, and a vector-borne pathogen. Genomic variations of BATV exist in different regions of the world, due to genetic reassortment. Whole-genome sequencing of any isolate is necessary for a phylogenetic analysis. In 1998, a BATV strain was isolated from an Anopheles philippines mosquito in Yunnan Province, China. This strain has not been found to infect any other host. We investigated BATV infection in cattle in Inner Mongolia, China and performed deep sequencing of the genome of the BATV isolate. FINDINGS: Ninety-five blood samples were collected from cattle in Inner Mongolia, China in 2012. The BATV infection rate was 2.1%. Previously, BATV strain NM/12 was isolated from two cattle in Inner Mongolia, China, and the whole genomic sequence of the strain has been available. We determined the complete genomic nucleotide sequences of the small (S), medium (M), and large (L) genome segments using bovine blood obtained in 2012, and the nucleotide homologies of these segments with those from GenBank were 88.7%-97%, 84%-95.4%, and 72.6%-95.8%, respectively. The deduced amino acid identities were 87.2-99.7%, 64.2-96.8%, and 81.1-98.6%. Phylogenetic analyses based on full-length genomic sequences indicated that the M and L segments, and a portion of the S segment, of NM/12 are most closely related to the BATV strains isolated in Asia. The S and M segments of NM/12 were independent of phylogenetic lineages. The L segment was the most closely related to Chittoor/IG-20217 (isolated in India), and distantly related to isolated strains in Italy. Nucleotide substitution rates in the nucleotide sequences that code for the nucleocapsid, envelope glycoprotein, and polymerase protein of NM/12 strain were 2.56%, 4.69%, and 4.21%, respectively, relative to the original strain of MM2222. CONCLUSION: A novel BATV NM/12 strain from bovine serum collected in Inner Mongolia was isolated from cattle in China for the first time. Our findings elucidate the evolutionary status of the BATV NM/12 strain among different orthobunyavirus strains and may provide some clues to prevent the emergence of BATV infection in cattle and humans.

Ag+ as a more effective elicitor for production of tanshinones than phenolic acids in Salvia miltiorrhiza hairy roots.

Phenolic acids and tanshinones are two groups of bioactive ingredients in Salvia miltiorrhiza Bunge. As a heavy metal elicitor, it has been reported that Ag+ can induce accumulations of both phenolic acids and tanshinones in S. miltiorrhiza hairy roots. In this study, the effects of Ag+ treatment on accumulations of six phenolic acids and four tanshinones in S. miltiorrhiza hairy roots were investigated. To further elucidate the molecular mechanism, expressions of key genes involved in the biosynthesis of these ingredients were also detected. The results showed that although the total phenolic acids content was almost not affected by Ag+, accumulations of rosmarinic acid (RA), caffeic acid and ferulic acid were significantly increased, while accumulations of salvianolic acid B (LAB), danshensu (DSU) and cinnamic acid were decreased. We speculate that LAB probably derived from the branch pathway of DSU biosynthesis. Contents of four tanshinones were enhanced by Ag+ and their accumulations were more sensitive to Ag+ than phenolic acids. Genes in the upstream biosynthetic pathways of these ingredients responded to Ag+ earlier than those in the downstream biosynthetic pathways. Ag+ probably induced the whole pathways, upregulated gene expressions from the upstream pathways to the downstream pathways, and finally resulted in the enhancement of ingredient production. Compared with phenolic acids, tanshinone production was more sensitive to Ag+ treatments. This study will help us understand how secondary metabolism in S. miltiorrhiza responds to elicitors and provide a reference for the improvement of the production of targeted compounds in the near future.

Lingdan: enhancing encoding of traditional Chinese medicine knowledge for clinical reasoning tasks with large language models.

OBJECTIVE: The recent surge in large language models (LLMs) across various fields has yet to be fully realized in traditional Chinese medicine (TCM). This study aims to bridge this gap by developing a large language model tailored to TCM knowledge, enhancing its performance and accuracy in clinical reasoning tasks such as diagnosis, treatment, and prescription recommendations. MATERIALS AND METHODS: This study harnessed a wide array of TCM data resources, including TCM ancient books, textbooks, and clinical data, to create 3 key datasets: the TCM Pre-trained Dataset, the Traditional Chinese Patent Medicine (TCPM) Question Answering Dataset, and the Spleen and Stomach Herbal Prescription Recommendation Dataset. These datasets underpinned the development of the Lingdan Pre-trained LLM and 2 specialized models: the Lingdan-TCPM-Chat Model, which uses a Chain-of-Thought process for symptom analysis and TCPM recommendation, and a Lingdan Prescription Recommendation model (Lingdan-PR) that proposes herbal prescriptions based on electronic medical records. RESULTS: The Lingdan-TCPM-Chat and the Lingdan-PR Model, fine-tuned on the Lingdan Pre-trained LLM, demonstrated state-of-the art performances for the tasks of TCM clinical knowledge answering and herbal prescription recommendation. Notably, Lingdan-PR outperformed all state-of-the-art baseline models, achieving an improvement of 18.39% in the Top@20 F1-score compared with the best baseline. CONCLUSION: This study marks a pivotal step in merging advanced LLMs with TCM, showcasing the potential of artificial intelligence to help improve clinical decision-making of medical diagnostics and treatment strategies. The success of the Lingdan Pre-trained LLM and its derivative models, Lingdan-TCPM-Chat and Lingdan-PR, not only revolutionizes TCM practices but also opens new avenues for the application of artificial intelligence in other specialized medical fields. Our project is available at https://github.com/TCMAI-BJTU/LingdanLLM.

Compound Danshen Dripping Pill inhibits hypercholesterolemia/atherosclerosis-induced heart failure in ApoE and LDLR dual deficient mice via multiple mechanisms.

Heart failure is the leading cause of death worldwide. Compound Danshen Dripping Pill (CDDP) or CDDP combined with simvastatin has been widely used to treat patients with myocardial infarction and other cardiovascular diseases in China. However, the effect of CDDP on hypercholesterolemia/atherosclerosis-induced heart failure is unknown. We constructed a new model of heart failure induced by hypercholesterolemia/atherosclerosis in apolipoprotein E (ApoE) and LDL receptor (LDLR) dual deficient (ApoE-/-LDLR-/-) mice and investigated the effect of CDDP or CDDP plus a low dose of simvastatin on the heart failure. CDDP or CDDP plus a low dose of simvastatin inhibited heart injury by multiple actions including anti-myocardial dysfunction and anti-fibrosis. Mechanistically, both Wnt and lysine-specific demethylase 4A (KDM4A) pathways were significantly activated in mice with heart injury. Conversely, CDDP or CDDP plus a low dose of simvastatin inhibited Wnt pathway by markedly up-regulating expression of Wnt inhibitors. While the anti-inflammation and anti-oxidative stress by CDDP were achieved by inhibiting KDM4A expression and activity. In addition, CDDP attenuated simvastatin-induced myolysis in skeletal muscle. Taken together, our study suggests that CDDP or CDDP plus a low dose of simvastatin can be an effective therapy to reduce hypercholesterolemia/atherosclerosis-induced heart failure.

Evidence for natural recombination between mink enteritis virus and canine parvovirus.

A virus was isolated from mink showing clinical and pathological signs of enteritis in China. This virus, designated MEV/LN-10, was identified as mink enteritis virus (MEV) based on its cytopathic effect in the feline F81 cell line, the hemagglutination (HA) and hemagglutination inhibition (HI) assay, electron microscopy (EM) and animal infection experiments. The complete viral genome was cloned and sequenced. Phylogenetic and recombination analyses on the complete MEV/LN-10 genome showed evidence of recombination between MEV and canine parvovirus (CPV). The genome was composed of the NS1 gene originating from CPV while the VP1 gene was of MEV origin. This is the first demonstration of recombination between a CPV and MEV in nature. Our findings not only provide valuable evidence indicating that recombination is an important genetic mechanism contributing to the variation and evolution of MEV, but also that heterogeneous recombination can occur in the feline parvovirus subspecies.

[Establishment and application of a Vero cell line stably expressing raccoon dog SLAM, the cellular receptor of canine distemper virus].

OBJECTIVE: The signaling lymphocyte activation molecule (SLAM, also known as CD150), is used as a cellular receptor by canine distemper virus (CDV). Wild-type strains of CDVs can be isolated and propagated efficiently in non-lymphoid cells expressing this protein. Our aim is to establish a Vero cells expressing raccoon dog SLAM (rSLAM) to efficiently isolate CDV from pathological samples. METHODS: A eukaryotic expression plasmid, pIRES2-EGFP-rSLAMhis, containing rSLAM gene fused with six histidine-coding sequence, EGFP gene, and neomycin resistance gene was constructed. After transfection with the plasmid, a stable cell line, Vero-rSLAM, was screened from Vero cells with the identification of EGFP reporter and G418 resistance. Three CD positive specimens from infected foxes and raccoon dogs were inoculated to Vero-rSLAM cells for CDV isolation. Foxes and raccoon dogs were inoculated subcutaneously LN (10)fl strain with 4 x 10(2.39)TCID50 dose to evaluate pathogenicity of CDV isolations. RESULTS: The rSLAMh fused gene was shown to transcript and express stably in Vero-rSLAM cells by RT-PCR and Immunohistochemistry assay. Three CDV strains were isolated successfully in Vero-rSLAM cells 36 -48 hours after inoculation with spleen or lung specimens from foxes and raccoon dogs with distemper. By contrast, no CDV was recovered from those CD positive specimens when Vero cells were used for virus isolation. Infected foxes and raccoon dogs with LN(10)f1 strain all showed typical CD symptoms and high mortality (2/3 for foxes and 3/3 for raccoon dogs) in 22 days post challenge. CONCLUSION: Our results indicate that Vero-rSLAM cells stably expressing raccoon dog SLAM are highly sensitive to CDV in clinical specimens and the CDV isolation can maintain high virulence to its host animals.